Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
- RNA used for tailing reactions should be purified prior to use and suspended in nuclease-free water. EDTA should not be present and the solution should be free of salts.
- RNase Inhibitor may be added to enhance the stability of the RNA in solution. If this is desired, 0.5 µl of a standard RNase inhibitor (such as RNase Inhibitor, Murine, NEB #M0314) can be added at the time of reaction set-up. The additional volume can be subtracted from the amount of H2O used in the reaction.
- Add the following components in the order specified:
- Incubate reaction at 37°C for 30 minutes.
- Stop the reaction by adding EDTA to a final concentration of 10 mM or directly proceeding to the cleanup step.
|RNA||1-10 µg in 15 µl nuclease free water|
|10X E. coli Poly(A) Polymerase Reaction Buffer||2 µl (1X)|
|ATP (10mM)||2 µl|
|E. coli Poly(A) Polymerase||1 µl|